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The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity. SOS mutagenesis is due to an error-prone mode of DNA replication expressed in wild-type Escherichia coli after exposure to UV light or to chemical agents that block DNA replication and induce the SOS response. In certain mutant strains in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity by which spontaneous mutation rates are elevated as much as 50-fold. In either case, SOS mutagenesis depends on the products of the recA, lexA, and umuDC genes. RecA and LexA proteins regulate the SOS response, and UmuDC proteins are believed to be essential for the actual mutagenic event (for reviews, see references 36 and 37).RecA protein has two essential roles in SOS mutagenesis. One is its regulatory role. DNA damage generates a signal that results in an altered form of RecA protein (RecA*). RecA* causes the proteolytic cleavage of LexA, the repressor of some 20 genes composing the SOS regulon, including recA itself and the umuDC operon (for reviews, see references 19 and 28). Amplification of UmuDC is necessary but not sufficient for SOS mutagenesis, as shown by the absence or weak expression of SOS mutator activity in recA+ strains carrying a lexA(Def) mutation that inactivates LexA. Such strains become strong mutators only if the recA+ allele is replaced by recA441 or recA730, which encode spontaneously activated RecA proteins, indicating that RecA* has an essential role in SOS mutagenesis other than its antirepressor function (la, 11, 38). It has recently been shown that RecA* promotes the proteolytic cleavage of UmuD protein both in vitro (5) and in vivo (32) and that only the larger COOH-terminal fragment (UmuD') is active in UV mutagenesis, whereas the unprocessed UmuD protein is not (27). SOS

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Mutation frequency decline (NWD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occtnng when protein sythesis translendy inhibited Immediately fter irrtin. MD is abolihed by mutations inthe uvrA, -B. or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate ofexcision but does not affect survival. Using an in vitro repair synthesis assay we found that although wild-type ells repair the ibed (template) strand frentially, mfd cells are incapable of d-pcfic repar. The iciency in strand-selective repair of ,*d-cell extract was cor d by adding highiy purified "transcription-repair coupling fator" to the reaction mixt. We conclude that mfd is, most likely, the gene encoding the tnscription-repai coupling factor.In recent years in vivo studies have shown that, in general, actively transcribing genes are repaired at a faster rate than the rest of the genome (1-3). In the majority of the cases gene-specific 'repair appears to be due to strand-specific repair-that is, in an. actively transcribing gene the template (transcribed) strand is repaired at such a high efficiency as to account for all ofthe gene-specific repair, whereas the coding (nontranscribed) strand is repaired 'at essentially the same rate as the rest of the genome (4, 5). Recently, we have developed an in vitro system (6, 7) capable of gene-and strand-specific repair and we have partially puified an Escherichia coli protein that confers strand specificity onto the E. coli nucleotide excision repair enzyme, (A)BC excinuclease. In this communication we describe the purification of the "transcription-repair coupling factor"' (TRCF) to nearhomogeneity and the preliminary identification of the coupling factor as the mfd gene product.MFD (mutation'frequency decline) is operationally defined as the rapid and irreversible decrease in the frequency of certain damage-induced suppressor mutations that occurs when protein synthesis is transiently inhibited immediately after irradiation (8)(9)(10)(11)(12) from' E. coli B/r and its mfd derivative were tested for strand-specific repair in vitro. We found that E. coli B/r, like E. coli K-12, was capable of strand-specific repair. In contrast, E. coli B/r mfd-extract was totally deficient in strand-specific repair. When we added the purified TRCF to the mutant cell extract it restoredthe strand-specific repair to the wild-type level. The most likely explanation of our data is that nfd encodes the TRCF. MATERIALS AND METHODS Cdls and.. E. coli K-12 derivatives AB1157 (wild type) and AB1886 (uvrA-) were used for making extractsfor routine repair synthesis assay and for purification of the TRCE, respectively. E. coli B/r derivative WU3610 (which is Leu-and Tyr-because of UAG and UAA mutations) and its derivative' WU3610-45 (mfd-1) are the strains that have frequently been used in studies on MFD (11). The plasmid pDR3274 (19) contains the uvrC gene under the strong tac promoter. Transcription from this promoter can be inhibited by rifampicin (Rif) ...

DNA REPAIR MECHANISMS IN ESCHERICHIA COLIThree repair mechanisms are now known in E. coli, each of which pro motes survival after exposure to ultraviolet light. Two of the three (photo-

Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC. Recovery of DNA synthesis after UV irradiation ("induced replisome reactivation," or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein [Khidhir, M. A., Casaregola, S. & Holland, I. B. (1985) Mol. Gen. Genet. 199, 133-140]. IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants. In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present. IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation. We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein. We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis.

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